Cellulose is the major structural component of higher plants and occurs naturally in almost pure form only in cotton fiber. It provides plant cells with high tensile strength helping them to resist mechanical stress and osmotic pressure. Cellulose is a linear polysaccharide of glucose residues connected by β-1,4 linkages. In nature, cellulose is usually associated with lignin together with hemicelluloses, such as xylans and glucomannans. Cellulolytic enzymes hydrolyze cellulose and are produced by a wide variety of bacteria and fungi. Cellulases are industrially important enzymes with a current annual market value of about 190 million U.S. $. In the textile industry, cellulases are used in denim finishing to create a fashionable stone washed appearance in denim cloths in a biostoning process, and they are also used, for instance, to clean fuzz and prevent formation of pills on the surface of cotton garments. In detergent industry cellulases are used to brighten colors and to prevent graying and pilling of garments. Cellulases are further used in food industry and animal feed manufacturing, and they have a great potential in the pulp and paper industry, for instance, in deinking to release ink from fiber surfaces and in improving pulp drainage. The wide spectrum of industrial uses for cellulases has established a need for commercial cellulase products containing different cellulase components and functioning optimally in different pH and temperature ranges.
The practical use of cellulases is hampered by the nature of the known cellulase compositions, which are often enzyme mixtures having a variety of activities and substrate specificities. For this reason, efforts have been made to obtain cellulases having only the desired activities. The unique properties of each cellulase make some more suitable for certain purposes than others. While the enzymes differ in a number of ways, one of the most important differences is the pH optimum. Neutral cellulases are most active in the pH range 6–8 and alkaline cellulases in the pH range 7.5–10, whereas acid cellulases, having the pH optimum at pH 4.5–5.5, show very low activity levels at higher pH values. Neutral and acid cellulases are especially useful in the textile industry. In fabric treatment cellulases attack the chains of cellulose molecules that form the cotton fibers, thereby affecting the characteristics of the fabric.
In textile industry “stone washed” look or an abraded look has been denim producers' interest in recent years. Traditional stone washing with pumice stones reduces the strength of fabric and burdens the laundering apparatuses. The trend has been towards enzymatic denim finishing processes and cellulases have replaced or are being used together with pumice stones to give the fabric its desired “worn” look. Controlled enzyme treatments result in less damage to the garments and machines and eliminate the need for disposal of stones.
Additionally, textile industry uses cellulases in biofinishing, i.e. to create permanent improvement of depilling and improved pilling resistance, cleared surface structure by reduced fuzz, improved textile handle, such as softness, smoothness and a silkier feel, improved drapability and brighter colors of the textile and improved moisture absorbability.
Cellulases applied in denim treatment are usually divided into two main groups: acid and neutral cellulases. Acid cellulases typically operate at pH 4.0–5.5 and the neutral cellulases in the range of pH 6–8. Acid cellulases used in biostoning mainly originate from Trichoderma reesei (sexual form Hypocrea jecorina) and the neutral cellulases come from a variety of fungi, including genera of Melanocarpus, Humicola, Myceliophthora, Fusarium, Acremonium, and Chrysosporium (Haakana et al. 2004). T. reesei enzymes include, e.g., cellulases from the glycoside family 5 (endoglucanase II, EGII), family 7 (cellobiohydrolase I, CBHI) and family 12 (endoglucanase III, EGIII; Ward et al. 1993), and the neutral cellulases, most often endoglucanases, from family 45 and family 7 (Henrissat, 1991; Henrissat and Bairoch, 1993, 1996).
Cellulases comprise a catalytic domain/core (CD) expressing cellulase activity. In addition to the catalytic domain the cellulase molecule may comprise one or more cellulose binding domains (CBDs), also named as carbohydrate binding domains/modules (CBD/CBM), which can be located either at the N- or C-terminus of the catalytic domain. CBDs have carbohydrate-binding activity and they mediate the binding of the cellulase to crystalline cellulose but have little or no effect on cellulase hydrolytic activity of the enzyme on soluble substrates. These two domains are typically connected via a flexible and highly glycosylated linker region.
Cellulases that attack primarily on the surface of the fiber are especially useful in stone washing of denim dyed with Indigo dye, as the dye is located on the surface of the fiber. When used to treat cotton fabric, acid cellulases generally require a shorter washing time than neutral cellulases. Acid cellulases are especially used in biofinishing (depilling) and also in denim treatment (biostoning).
Endoglucanases (EGs) in connection of the present invention mean enzymes classified as E.C. 3.2.1.4 and are one of the three types of cellulases generally needed for the biological conversion of cellulose to glucose. Endoglucanases cut internal beta-1,4-glucosidic bonds, whereas cellobiohydrolases cut the disaccharide cellobiose from the end of the cellulose polymer chain and beta-1,4-glucosidases hydrolyze the cellobiose and other short cello-oligosaccharides to glucose. Some naturally occurring endoglucanases have a cellulose-binding domain (CBD), while others do not.
Also endoglucanases are widely used in textile, detergent, and pulp and paper industry. For instance, endoglucanases of the cel45 family (EGs fam 45) are described, e.g., in U.S. Pat. No. 6,001,639, which describes enzymes having endoglucanase activity and having two conserved amino acid sequences. Uses in textile, detergent, and pulp and paper applications are generally discussed and treating of lignocellulosic material is mentioned. WO 2004/053039 is directed to detergent applications of endoglucanases. U.S. Pat. No. 5,958,082 discloses the use of endoglucanase, especially from Thielavia terrestris in textile applications providing stoned washed or abraded look of twill jeans. EP 0495258 relates to detergent compositions containing Humicola cellulase. U.S. Pat. No. 5,948,672 describes a cellulase preparation containing endoglucanase, especially from Humicola and its use in textile and pulp applications.
EG:s and EG-enriched compositions and concentrates are also commercially available.
However, there is a continuous need for improved cellulases, including endoglucanases that are more efficient in fabric treatment and in other fields, where cellulases traditionally are used. In particular, there is a continuous need for more efficient cellulases to improve the process economics.
The present invention aims to meet this need.